Attempts to combat HIV have been hampered due to the virus's ability to rapidly mutate and produce genetic variants that can circumvent the immune response and resist drug therapy. The tremendous genetic diversity of HIV has greatly complicated the already daunting task of producing an effective vaccine for a virus capable of spreading through cell to cell contact and concealing its genetic material in the proviral state within populations of dormant reservoir cells. Highly Active Anti-Retroviral Therapy (HAART) has been a powerful tool for treating HIV patients but it has not effectively reached the most vulnerable populations, while resistance to many of the drugs used in HAART has inevitably emerged. Alternative approaches will be necessary in the future to help control and overcome HIV infections and AIDS. This application aims to contribute to these goals by more clearly defining the mechanism of HIV reverse transcriptase (RT) fidelity and examining the effect of physiological conditions on fidelity and the utilization of commonly used RT inhibitors. Experiments that show how RT biochemistry occurs under cellular conditions indicate that current literature on RT fidelity and interactions with drugs like AZT and ddC is misleading. Results from the proposed experiments will more clearly define how RT works in the cell making it easier to develop and evaluate potential new drugs. A novel approach for crystallization of HIV RT using primer-template mimics that bind very tightly to RT is also demonstrated in collaboration with Dr. Eddy Arnold's group at Rutgers. This method allows for the first time, rapid crystallization of RT in the absence of cross-linking and leads to the formation of catalytically active crystals that can be treated with RT inhibitors to investigate the structure o the RT-inhibitor complexes. Information can be used for structure-based design of novel RT inhibitors. Aptamers (nucleic acids that bind extremely tightly to target proteins) can potentially be used for diagnostic and therapeutic applications, such as replacements of antibodies in biochemical assays (e.g. ELISA), utilization as biosensors, as tools for studying virus molecular biology, and as models for antiviral drugs. Aptamers specific to HIV and other retroviruses have been shown to inhibit virus replication in cell and animal models. In this proposal, a new class of aptamers to HIV RT that are composed of Xeno nucleic acids (XNA) will be tested for viral inhibition along with the novel primer-template mimicking aptamers used for crystallization above. XNAs are composed of nucleotide mimics that resemble normal nucleotides but contain unique chemical and structural modification that differentiate them from normal nucleotides. XNA aptamers are unique because the unnatural structure of the nucleoside is less susceptible to degradative enzymes and other harsh conditions, while they are also less likely to be recognized by the innate immune system than current RNA and DNA aptamers.